An experimental framework to assess biomolecular condensates in bacteria

High-resolution imaging of biomolecular condensates in living cells is essential for correlating their properties to those observed through in vitro assays. However, such experiments are limited in bacteria due to resolution limitations. Here we present an experimental framework that probes the formation, reversibility, and dynamics of condensate-forming proteins in Escherichia coli as a means to determine the nature of biomolecular condensates in bacteria. We demonstrate that condensates form after passing a threshold concentration, maintain a soluble fraction, dissolve upon shifts in temperature and concentration, and exhibit dynamics consistent with internal rearrangement and exchange between condensed and soluble fractions. We also discover that an established marker for insoluble protein aggregates, IbpA, has different colocalization patterns with bacterial condensates and aggregates, demonstrating its potential applicability as a reporter to differentiate the two in vivo. Overall, this framework provides a generalizable, accessible, and rigorous set of experiments to probe the nature of biomolecular condensates on the sub-micron scale in bacterial cells.


Figure S1 .
Figure S1.Overexpression of mNG-McdB in E. coli BL21.a. mNG-McdB forms foci.The phase contrast channel is shown in gray and the mNG channel is in green.Images are representative of three biological replicates.White arrows highlight cells where McdB foci wet to the inner membrane inducing cell curvature.Scale bars: 5 µm.b. mNG-McdB foci are nucleoid-excluded.DAPI stain is shown in magenta.Scale bar: 1 µm.c.Effects of changing cell volume on focus stability.E. coli cell volume was increased by treating cells with the MreB inhibitor, A22 (10 µg/mL).Images were taken at 6 h post-treatment, when rod-shaped cells transitioned their morphology into round cells.Scale bar: 5 µm.d.Quantification of cells shown in (c).Solid lines and shading represent the mean condensation coefficient of a representative cell population (n > 10 cells) and its 95% confidence interval.An intensity threshold of I = 0.5 was used; a condensation coefficient of 50 is equivalent to a homogenous pixel intensity distribution.e. Localized lysis of the cell.One cell pole was lysed using a high-intensity laser, focused within the indicated magenta circle.Representative frames show solubilization of the opposing focus and shift of fluorescent signal to the opposite end.Scale bar: 1 µm.f.Quantification of three representative cells (n = 3) for each protein shown in (e).Solid lines and shading represent the mean condensation of three representative cells and the standard deviation.An intensity threshold of I = 0.1 was used.

Figure S2 .
Figure S2.Expression of mCherry fusion proteins.E. coli MG1665 cells of indicated strains were induced with 1 mM IPTG for 2 h.a. Immunoblot analysis of mCherry fusion proteins.Representative immunoblots are shown.Red box shows the band at the mCherry size (28 kDa).Similar results were observed in three biological replicates.b.Cleavage level of mCherry fusion proteins.In each lane, the intensity of the band at the mCherry size was quantified and normalized to the total intensity of the respective lane.Error bars represent the standard deviation from the three biological replicates (n = 3).

Figure S3 .
Figure S3.mCherry fusion proteins form cellular foci.Representative images of indicated mCherry fusion protein foci in E. coli from 0 to 5 h post-induction.Phase contrast (blue) and mCherry (green) channels were merged.Images are representative of three biological replicates (n = 3).Scale bar: 1 m.

Figure S4 .
Figure S4.Electron-multiplying charge-coupled detector (EMCCD) calibration for photon counting.a. Signal variance versus signal average for different camera integration times.The data points correspond to the following camera integration times: 10, 20, 40, 80, 160, and 320 ms.The dashed red line is the linear regression.b.Electron multiplication conversion plot.The dashed red line is the linear regression constrained through the origin.c.Distribution of number of detected photons per localization per imaging frame for mCherry-McdB.Signal histogram was fit to a gamma distribution, determining a peak of 91.1 photons per localization at a 40-ms camera integration time.35800 localizations were used to build the histogram.

Figure S5 .
Figure S5.Normalized pixel intensity histograms and condensation coefficients of mCherry fusion proteins.a-f.Normalized pixel intensity histograms for cells with no detected focus (left) and a detected focus (right).Black lines represent individual cells.The red dashed line and shading are the average and standard deviation across cells for each plot.g.Quantification of condensation coefficients.Condensation coefficient plots for cells with a detected focus and threshold values of I = 0.5 and 0.7.Data points correspond to individual cells.The curves next to the scatter plots were obtained via kernel density estimation.The shaded region represents the measurement range for cells expressing a uniform mCherry signal.Analysis was done on N > 900 cells for each fusion over three biological replicates.

Figure
Figure S6.mCherry-McdB sol condenses by repulsive interactions with the nucleoid.a. Representative images of mCherry-McdB foci in E. coli at 4 h post-induction (top).Cells were treated with ciprofloxacin (50 mM) for nucleoid compaction and stained with DAPI (2 µM) for nucleoid visualization (bottom).mCherry channel (green), DAPI (magenta), and merged images overlaid with Phase contrast (blue) are shown.White arrows highlight the mCherry-McdB foci that remained after nucleoid compaction.Magenta bars highlight the size of the nucleoid before and after ciprofloxacin treatment.b.Normalized profile lines along the normalized lengths of the long axes of four representative cells (n = 4) from the population shown in (a).Green and magenta lines represent the mCherry and DAPI signals, respectively.Solid lines and dashed lines represent the signal before and after ciprofloxacin treatment, respectively.c.Same as in (a) for mCherry-McdB sol cells.Green bars indicate the length of the mCherry-McdB sol foci before and after ciprofloxacin treatment.d.Same as in (b) for cells from the population shown in (c).Images are representative of three biological replicates.Scale bar: 1 µm.

Figure S7 .
Figure S7.Condensate reversibility through cell growth, division, and osmolarity shift.a. Generational dilution dissolves phase-separated foci.The phase contrast channel (blue) and the mCherry channel (green) are merged for a-c.White arrows demarcate the same cellular location of the same focus over time.Blank arrows demarcate the cellular position now absent of a focus.Images are representative of four biological replicates.Scale bars: 2 µm.b.Cell elongation dissolves phase-separated foci.As in (a), except cells were treated with 10 µg/ml cephalexin to block cell division prior to spotting on agar pads.c.Cells osmotically shocked with 300 mM NaCl.The first row of panels shows cells treated cells prewash; 15 and 30 min are cells post-wash.Images are representative of three biological replicates.d.Percent of cells with a fluorescent focus for osmotically shocked cells shown in (c) and for untreated control cells from three biological replicates.Bars indicate the standard deviation across the replicates.N > 50 cells for each protein at each time point per replicate.

Figure S8 .
Figure S8.Diffusion coefficients of PAmCherry fusion proteins.Normalized log apparent diffusion coefficient histograms and two-component Gaussian mixture fit.The solid black line corresponds to the two-component Gaussian fit.The dashed black lines represent the Gaussian fit of each component which we refer to as Dapp,slow and Dapp,fast.N indicates the number of trajectories analyzed for each protein per condition.

Figure S9 .
Figure S9.The nature of IbpA chaperone association with foci informs their material state in vivo.Wide-field fluorescence images of mCherry fusion foci (magenta) and IbpA-msfGFP foci (green).Phase Contrast is blue in the merge.Images are representative of three biological replicates.Scale bar: 2 µm.b.Quantification of the association of the foci of mCherry fusions and IbpA-msfGFP.Data points correspond to the ratio of the mean full-width at half maximum (FWHM) for the two channels (FWHMIbpA/FWHMmCherry) for the sum projections of technical replicates (cI agg : 5, PopTag SL : 7, PopTag LL : 4, McdB: 5) over three independent experiments.n > 30 foci for each protein per technical replicate.Black lines represent the mean ratio and the error bars represent the standard deviation.***p < 0.001 by Welch's t-test.